The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100734
ISSN1083-351X
AutoresSara Osman, Eusra Mohammad, Michael Lidschreiber, Alexandra Stüetzer, Fanni Laura Bazsó, Kerstin C. Maier, Henning Urlaub, Patrick Cramer,
Tópico(s)RNA Research and Splicing
ResumoThe Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro. We further showed that the Cdk8 kinase activity of the CKM weakened CKM–cMed interaction, thereby facilitating dissociation of the CKM and enabling mediator to bind the PIC in order to stimulate transcription initiation. Finally, we report that the kinase activity of Cdk8 is required for gene activation during the stressful condition of heat shock in vivo but not under steady-state growth conditions. Based on these results, we propose a model in which the CKM negatively regulates mediator function at upstream-activating sequences by preventing mediator binding to the PIC at the gene promoter. However, during gene activation in response to stress, the Cdk8 kinase activity of the CKM may release mediator and allow its binding to the PIC, thereby accounting for the positive function of CKM. This may impart improved adaptability to stress by allowing a rapid transcriptional response to environmental changes, and we speculate that a similar mechanism in metazoans may allow the precise timing of developmental transcription programs. The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro. We further showed that the Cdk8 kinase activity of the CKM weakened CKM–cMed interaction, thereby facilitating dissociation of the CKM and enabling mediator to bind the PIC in order to stimulate transcription initiation. Finally, we report that the kinase activity of Cdk8 is required for gene activation during the stressful condition of heat shock in vivo but not under steady-state growth conditions. Based on these results, we propose a model in which the CKM negatively regulates mediator function at upstream-activating sequences by preventing mediator binding to the PIC at the gene promoter. However, during gene activation in response to stress, the Cdk8 kinase activity of the CKM may release mediator and allow its binding to the PIC, thereby accounting for the positive function of CKM. This may impart improved adaptability to stress by allowing a rapid transcriptional response to environmental changes, and we speculate that a similar mechanism in metazoans may allow the precise timing of developmental transcription programs. Transcription of protein-coding genes begins when RNA polymerase II (pol II) and the general transcription factors (TFs)—IIA, IIB, IID (or the TATA box–binding protein [TBP]), IIE, IIF, and IIH—assemble at gene promoters to form a preinitiation complex (PIC). The PIC bends and unwinds promoter DNA and positions the pol II active center at the transcription start site (TSS) for initiation of DNA-templated RNA synthesis. Whereas PICs across different gene promoters are likely to be similar, diversification of transcriptional outputs is mainly achieved by binding of gene-specific TFs to upstream activation sequences (UASs) in yeast or to enhancers in metazoan cells. TFs that are bound to UASs or enhancers communicate their signals through coactivator complexes such as mediator and the Spt-Ada-Gcn5 acetyltransferase complex. These coactivators are large and modular complexes providing a plethora of possibilities to accommodate interactions with hundreds of different gene-specific TFs (1Hahn S. Young E.T. Transcriptional regulation in Saccharomyces cerevisiae: Transcription factor regulation and function, mechanisms of initiation, and roles of activators and coactivators.Genetics. 2011; 189: 705-736Crossref PubMed Scopus (198) Google Scholar). Coactivators may facilitate chromatin accessibility, modulate the frequency of PIC formation events, or stabilize PICs to favor their formation and/or dissociation and release of pol II into productive transcription. The mediator complex is a general coactivator and has a molecular weight of over 1 MDa. Mediator is composed of 25 subunits in yeast and 30 subunits in human (2Verger A. Monté D. Villeret V. Twenty years of mediator complex structural studies.Biochem. Soc. Trans. 2019; 47: 399-410Crossref PubMed Scopus (22) Google Scholar). Early studies of mediator structure identified four structural modules called the head, middle, tail, and Cdk8 kinase module (CKM) (3Asturias F.J. Jiang Y.W. Myers L.C. Gustafsson C.M. Kornberg R.D. Conserved structures of mediator and RNA polymerase II holoenzyme.Science. 1999; 283: 985-987Crossref PubMed Scopus (198) Google Scholar, 4Dotson M.R. Yuan C.X. Roeder R.G. Myers L.C. Gustafsson C.M. Jiang Y.W. Li Y. Kornberg R.D. Asturias F.J. Structural organization of yeast and mammalian mediator complexes.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14307-14310Crossref PubMed Scopus (151) Google Scholar). The essential head and middle modules form the core mediator (cMed). Structures of cMed in isolation (5Nozawa K. Schneider T.R. Cramer P. Core mediator structure at 3.4 Å extends model of transcription initiation complex.Nature. 2017; 545: 248Crossref PubMed Scopus (68) Google Scholar, 6Tsai K.L. Yu X. Gopalan S. Chao T.C. Zhang Y. Florens L. Washburn M.P. Murakami K. Conaway R.C. Conaway J.W. Asturias F.J. Mediator structure and rearrangements required for holoenzyme formation.Nature. 2017; 544: 196-201Crossref PubMed Scopus (81) Google Scholar) and interacting with pol II complexes (6Tsai K.L. Yu X. Gopalan S. Chao T.C. Zhang Y. Florens L. Washburn M.P. Murakami K. Conaway R.C. Conaway J.W. Asturias F.J. Mediator structure and rearrangements required for holoenzyme formation.Nature. 2017; 544: 196-201Crossref PubMed Scopus (81) Google Scholar, 7Plaschka C. Larivière L. Wenzeck L. Seizl M. Hemann M. Tegunov D. Petrotchenko E.V. Borchers C.H. Baumeister W. Herzog F. Villa E. Cramer P. Architecture of the RNA polymerase II–mediator core initiation complex.Nature. 2015; 518: 376-380Crossref PubMed Scopus (188) Google Scholar, 8Schilbach S. Hantsche M. Tegunov D. Dienemann C. Wigge C. Urlaub H. Cramer P. Structures of transcription pre-initiation complex with TFIIH and mediator.Nature. 2017; 551: 204-209Crossref PubMed Scopus (123) Google Scholar) have been determined. Recently, the structure of mediator including the head, middle, and tail modules from mouse was also reported (9Zhao H. Young N. Kalchschmidt J. Lieberman J. El Khattabi L. Casellas R. Asturias F.J. Structure of mammalian mediator complex reveals tail module architecture and interaction with a conserved core.Nat. Commun. 2021; 12: 1-12Crossref PubMed Scopus (13) Google Scholar). cMed is composed of a head module with two jaws, with a neck, spine, and arm, linking it to the middle module, which extends in an arch forming a knob and hook. cMed interacts with pol II in the PIC, forming three contacts (7Plaschka C. Larivière L. Wenzeck L. Seizl M. Hemann M. Tegunov D. Petrotchenko E.V. Borchers C.H. Baumeister W. Herzog F. Villa E. Cramer P. Architecture of the RNA polymerase II–mediator core initiation complex.Nature. 2015; 518: 376-380Crossref PubMed Scopus (188) Google Scholar). First, the arm/spine of the cMed head module contacts the pol II Rpb4–Rpb7 stalk. Second, the moveable jaw of the cMed head module contacts the pol II dock. Third, there is a transient interaction between the mobile plank domain of the cMed middle module and the pol II foot region. Recently, structures of human mediator–PIC complexes became available showing similar contacts (10Abdella R. Talyzina A. Chen S. Inouye C. Tjian R. He Y. Structure of the human mediator-bound transcription preinitiation complex.Science. 2021; 372: 52-56Crossref PubMed Scopus (26) Google Scholar, 11Rengachari S. Schilbach S. Aibara S. Dienemann C. Cramer P. Structure of human mediator-RNA polymerase II transcription pre-initiation complex.bioRxiv. 2021; ([preprint])https://doi.org/10.1101/2021.03.11.435010Crossref Scopus (0) Google Scholar). The CKM is present in a subpopulation of mediator complexes in cells (12Andrau J.-C. van de Pasch L. Lijnzaad P. Bijma T. Koerkamp M.G. van de Peppel J. Werner M. Holstege F.C.P. Genome-wide location of the coactivator mediator: Binding without activation and transient Cdk8 interaction on DNA.Mol. Cell. 2006; 22: 179-192Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 13Taatjes D.J. Näär A.M. Andel F. Nogales E. Tjian R. Structure, function, and activator-induced conformations of the CRSP coactivator.Science. 2002; 295: 1058-1062Crossref PubMed Scopus (207) Google Scholar), consistent with its dissociable nature. The CKM comprises the four subunits Med12, Med13, Cdk8, and cyclin C and is very large, roughly the same size as cMed (14Borggrefe T. Davis R. Erdjument-Bromage H. Tempst P. Kornberg R.D. A complex of the Srb8,-9,-10, and-11 transcriptional regulatory proteins from yeast.J. Biol. Chem. 2002; 277: 44202-44207Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Whereas the head, middle, and tail modules of mediator are present at both promoters and upstream regulatory sequences (15El Khattabi L. Zhao H. Kalchschmidt J. Young N. Jung S. Van Blerkom P. Kieffer-Kwon P. Kieffer-Kwon K.-R. Park S. Wang X. A pliable mediator acts as a functional rather than an architectural bridge between promoters and enhancers.Cell. 2019; 178: 1145-1158.e1120Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 16Fan X. Chou D.M. Struhl K. Activator-specific recruitment of mediator in vivo.Nat. Struct. Mol. Biol. 2006; 13: 117Crossref PubMed Scopus (76) Google Scholar, 17Fan X. Struhl K. Where does mediator bind in vivo?.PLoS One. 2009; 4e5029Crossref PubMed Scopus (52) Google Scholar, 18Jeronimo C. Langelier M.-F. Bataille A.R. Pascal J.M. Pugh B.F. Robert F. Tail and kinase modules differently regulate core mediator recruitment and function in vivo.Mol. Cell. 2016; 64: 455-466Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Petrenko N. Jin Y. Wong K.H. Struhl K. Mediator undergoes a compositional change during transcriptional activation.Mol. Cell. 2016; 64: 443-454Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), the CKM is present only at upstream sequences but not at promoters (18Jeronimo C. Langelier M.-F. Bataille A.R. Pascal J.M. Pugh B.F. Robert F. Tail and kinase modules differently regulate core mediator recruitment and function in vivo.Mol. Cell. 2016; 64: 455-466Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Petrenko N. Jin Y. Wong K.H. Struhl K. Mediator undergoes a compositional change during transcriptional activation.Mol. Cell. 2016; 64: 443-454Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The Cdk8 kinase phosphorylates the pol II C-terminal repeat domain (CTD) (20Hengartner C.J. Myer V.E. Liao S.-M. Wilson C.J. Koh S.S. Young R.A. Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases.Mol. Cell. 1998; 2: 43-53Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) and various gene-specific TFs resulting in both negative and positive effects on gene transcription (21Chen M. Liang J. Ji H. Yang Z. Altilia S. Hu B. Schronce A. McDermott M.S.J. Schools G.P. Lim C.-U. Oliver D. Shtutman M.S. Lu T. Stark G.R. Porter D.C. et al.CDK8/19 mediator kinases potentiate induction of transcription by NFκB.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 10208-10213Crossref PubMed Scopus (44) Google Scholar, 22Hirst M. Kobor M.S. Kuriakose N. Greenblatt J. Sadowski I. GAL4 is regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent protein kinase SRB10/CDK8.Mol. Cell. 1999; 3: 673-678Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 23Lenssen E. Azzouz N. Michel A. Landrieux E. Collart M.A. The Ccr4-not complex regulates Skn7 through Srb10 kinase.Eukaryot. Cell. 2007; 6: 2251-2259Crossref PubMed Scopus (21) Google Scholar, 24Liu Y. Kung C. Fishburn J. Ansari A.Z. Shokat K.M. Hahn S. Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex.Mol. Cell. Biol. 2004; 24: 1721-1735Crossref PubMed Scopus (143) Google Scholar, 25Rohde J.R. Trinh J. Sadowski I. Multiple signals regulate GALTranscription in yeast.Mol. Cell. Biol. 2000; 20: 3880-3886Crossref PubMed Scopus (57) Google Scholar, 26Sadowski I. Costa C. Dhanawansa R. Phosphorylation of Ga14p at a single C-terminal residue is necessary for galactose-inducible transcription.Mol. Cell. Biol. 1996; 16: 4879-4887Crossref PubMed Google Scholar, 27Sadowski I. Niedbala D. Wood K. Ptashne M. GAL4 is phosphorylated as a consequence of transcriptional activation.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10510-10514Crossref PubMed Scopus (54) Google Scholar, 28Steinparzer I. Sedlyarov V. Rubin J.D. Eislmayr K. Galbraith M.D. Levandowski C.B. Vcelkova T. Sneezum L. Wascher F. Amman F. Transcriptional responses to IFN-γ require mediator kinase-dependent pause release and mechanistically distinct CDK8 and CDK19 functions.Mol. Cell. 2019; 76: 485-499.e488Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 29Vincent O. Kuchin S. Hong S.-P. Townley R. Vyas V.K. Carlson M. Interaction of the Srb10 kinase with Sip4, a transcriptional activator of gluconeogenic genes in Saccharomyces cerevisiae.Mol. Cell. Biol. 2001; 21: 5790-5796Crossref PubMed Scopus (67) Google Scholar). Cdk8 phosphorylation increases the turnover of some TFs (30Chi Y. Huddleston M.J. Zhang X. Young R.A. Annan R.S. Carr S.A. Deshaies R.J. Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase.Genes Dev. 2001; 15: 1078-1092Crossref PubMed Scopus (256) Google Scholar, 31Nelson C. Goto S. Lund K. Hung W. Sadowski I. Srb10/Cdk8 regulates yeast filamentous growth by phosphorylating the transcription factor Ste12.Nature. 2003; 421: 187Crossref PubMed Scopus (123) Google Scholar, 32Raithatha S. Su T.-C. Lourenco P. Goto S. Sadowski I. Cdk8 regulates stability of the transcription factor Phd1 to control pseudohyphal differentiation of Saccharomyces cerevisiae.Mol. Cell. Biol. 2012; 32: 664-674Crossref PubMed Scopus (30) Google Scholar) but is also required for the activity of other factors (22Hirst M. Kobor M.S. Kuriakose N. Greenblatt J. Sadowski I. GAL4 is regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent protein kinase SRB10/CDK8.Mol. Cell. 1999; 3: 673-678Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 25Rohde J.R. Trinh J. Sadowski I. Multiple signals regulate GALTranscription in yeast.Mol. Cell. Biol. 2000; 20: 3880-3886Crossref PubMed Scopus (57) Google Scholar, 26Sadowski I. Costa C. Dhanawansa R. Phosphorylation of Ga14p at a single C-terminal residue is necessary for galactose-inducible transcription.Mol. Cell. Biol. 1996; 16: 4879-4887Crossref PubMed Google Scholar, 27Sadowski I. Niedbala D. Wood K. Ptashne M. GAL4 is phosphorylated as a consequence of transcriptional activation.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10510-10514Crossref PubMed Scopus (54) Google Scholar, 33Ansari A.Z. Koh S.S. Zaman Z. Bongards C. Lehming N. Young R.A. Ptashne M. Transcriptional activating regions target a cyclin-dependent kinase.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14706-14709Crossref PubMed Scopus (48) Google Scholar, 34Liao S.-M. Zhang J. Jeffery D.A. Koleske A.J. Thompson C.M. Chao D.M. Viljoen M. van Vuuren H.J. Young R.A. A kinase–cyclin pair in the RNA polymerase II holoenzyme.Nature. 1995; 374: 193Crossref PubMed Scopus (362) Google Scholar). CKM activity stimulates transcription of genes in stress and signal response networks (21Chen M. Liang J. Ji H. Yang Z. Altilia S. Hu B. Schronce A. McDermott M.S.J. Schools G.P. Lim C.-U. Oliver D. Shtutman M.S. Lu T. Stark G.R. Porter D.C. et al.CDK8/19 mediator kinases potentiate induction of transcription by NFκB.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 10208-10213Crossref PubMed Scopus (44) Google Scholar, 35Galbraith M.D. Allen M.A. Bensard C.L. Wang X. Schwinn M.K. Qin B. Long H.W. Daniels D.L. Hahn W.C. Dowell R.D. HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia.Cell. 2013; 153: 1327-1339Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 36Galbraith M.D. Andrysik Z. Pandey A. Hoh M. Bonner E.A. Hill A.A. Sullivan K.D. Espinosa J.M. CDK8 kinase activity promotes glycolysis.Cell Rep. 2017; 21: 1495-1506Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and its dysregulation has been implicated in over 100 different human cancers (37Clark A.D. Oldenbroek M. Boyer T.G. Mediator kinase module and human tumorigenesis.Crit. Rev. Biochem. Mol. Biol. 2015; 50: 393-426PubMed Google Scholar, 38Firestein R. Hahn W.C. Revving the throttle on an oncogene: CDK8 takes the driver seat.Cancer Res. 2009; 69: 7899-7901Crossref PubMed Scopus (35) Google Scholar, 39Philip S. Kumarasiri M. Teo T. Yu M. Wang S. Cyclin-dependent kinase 8: A new hope in targeted cancer therapy?.J. Med. Chem. 2018; 61: 5073-5092Crossref PubMed Scopus (50) Google Scholar). The CKM was proposed to sterically inhibit mediator from interacting with pol II at promoters (40Elmlund H. Baraznenok V. Lindahl M. Samuelsen C.O. Koeck P.J. Holmberg S. Hebert H. Gustafsson C.M. The cyclin-dependent kinase 8 module sterically blocks mediator interactions with RNA polymerase II.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15788-15793Crossref PubMed Scopus (149) Google Scholar, 41Tsai K.L. Sato S. Tomomori-Sato C. Conaway R.C. Conaway J.W. Asturias F.J. A conserved mediator-CDK8 kinase module association regulates mediator-RNA polymerase II interaction.Nat. Struct. Mol. Biol. 2013; 20: 611-619Crossref PubMed Scopus (139) Google Scholar), independently of its kinase activity (42Knuesel M.T. Meyer K.D. Bernecky C. Taatjes D.J. The human CDK8 subcomplex is a molecular switch that controls mediator coactivator function.Genes Dev. 2009; 23: 439-451Crossref PubMed Scopus (234) Google Scholar), but this has not been shown directly in a highly defined biochemical system. Also, it is not understood how the effects of Cdk8 kinase activity on gene expression relate to changes in the interactions between mediator, CKM, and the PIC. Here, we reconstitute an in vitro system to show that the CKM sterically inhibits cMed binding to the PIC, and that CKM-dependent phosphorylation releases this steric inhibition. Our data show that while CKM sterically represses transcription, its Cdk8 kinase activity releases this repression and enables gene activation. A hurdle toward the biochemical characterization of the CKM was obtaining the complex in sufficient quality and quantity. We established a method for heterologous coexpression of the complete yeast CKM (Fig. 1A) from a single vector in insect cells using a baculovirus-based expression system and its subsequent affinity purification (see Experimental procedures section). We prepared two variants of the CKM module. The first variant contains the catalytically active Cdk8 subunit (CKM(A)), and the second variant contains the kinase-dead Cdk8 mutation D286A (CKM(KD)). Both variants behaved similarly in biochemical purification (Fig. S1). Purified CKM(A) readily phosphorylated the pol II CTD on both serine-2 and serine-5 residues (Fig. 1C). These results show that our recombinant CKM module was active as a kinase on a natural substrate. We next performed negative stain EM on CKM(KD). We fixed the complex with glutaraldehyde during sucrose gradient ultracentrifugation according to the GraFix protocol (43Kastner B. Fischer N. Golas M.M. Sander B. Dube P. Boehringer D. Hartmuth K. Deckert J. Hauer F. Wolf E. GraFix: Sample preparation for single-particle electron cryomicroscopy.Nat. Methods. 2008; 5: 53Crossref PubMed Scopus (298) Google Scholar) and incubated complex-containing fractions on carbon foil–coated EM grids, stained with uranyl formate. EM analysis showed homogeneous particles, evenly distributed over the surface of the grid (Fig. 1D). 2D classes calculated from roughly 60,000 particles showed that the majority of the particles were intact, confirming the homogeneity and quality of the sample. An ab initio 3D model generated using CryoSparc SPA Cryo-EM Software Systems (44Punjani A. Rubinstein J.L. Fleet D.J. Brubaker M.A. cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination.Nat. Methods. 2017; 14: 290Crossref PubMed Scopus (1394) Google Scholar) showed an elongated volume with three prominent wedges forming an overall shape resembling the capital letter "E" (Fig. 1E), which is consistent with the low-resolution cryo-EM densities previously reported for endogenously purified CKM (41Tsai K.L. Sato S. Tomomori-Sato C. Conaway R.C. Conaway J.W. Asturias F.J. A conserved mediator-CDK8 kinase module association regulates mediator-RNA polymerase II interaction.Nat. Struct. Mol. Biol. 2013; 20: 611-619Crossref PubMed Scopus (139) Google Scholar, 45Wang X. Wang J. Ding Z. Ji J. Sun Q. Cai G. Structural flexibility and functional interaction of mediator Cdk8 module.Protein Cell. 2013; 4: 911-920Crossref PubMed Scopus (17) Google Scholar). While this article was under revision, a higher resolution CKM structure was also reported (46Li Y.-C. Chao T.-C. Kim H.J. Cholko T. Chen S.-F. Li G. Snyder L. Nakanishi K. Chang C.-e. Murakami K. Structure and noncanonical Cdk8 activation mechanism within an Argonaute-containing mediator kinase module.Sci. Adv. 2021; 7eabd4484Crossref PubMed Scopus (8) Google Scholar). We next tested whether the CKM can bind to the 16-subunit cMed, which we produced in recombinant form and at high homogeneity as described (7Plaschka C. Larivière L. Wenzeck L. Seizl M. Hemann M. Tegunov D. Petrotchenko E.V. Borchers C.H. Baumeister W. Herzog F. Villa E. Cramer P. Architecture of the RNA polymerase II–mediator core initiation complex.Nature. 2015; 518: 376-380Crossref PubMed Scopus (188) Google Scholar, 8Schilbach S. Hantsche M. Tegunov D. Dienemann C. Wigge C. Urlaub H. Cramer P. Structures of transcription pre-initiation complex with TFIIH and mediator.Nature. 2017; 551: 204-209Crossref PubMed Scopus (123) Google Scholar). Binding of the CKM to cMed was evident by a shift in the complex-containing fractions to higher density fractions upon sucrose density gradient ultracentrifugation (Fig. 1F). These results show that recombinant CKM binds cMed in vitro. Having purified CKM and cMed at hand, we asked whether these complexes could bind to the pol II PIC. To test this, we set up an immobilized template assay (ITA) using a DNA scaffold containing a promoter sequence modeled on the yeast HIS4 promoter, with a TATA box and a TSS, as a platform for PIC assembly. Biotinylation on the DNA 3' end allowed immobilization of the template on streptavidin beads, and an EcoRV restriction site downstream of the promoter sequence allowed elution by endonuclease cleavage. We could readily form PICs on the immobilized promoter template (Fig. 2A) providing a positive control. We then incubated CKM(A) or CKM(KD) with the immobilized template, purified cMed, and purified PIC components pol II, TFIIA, TFIIB, TFIIE, TFIIF, and TBP. We excluded TFIIH to reduce the complexity of the system to a single catalytic activity being tested. The restriction digestion elutions were analyzed by SDS-PAGE to investigate what was bound to promoter DNA together with the PIC (Fig. 2A). In the presence of PIC components, cMed bound stoichiometrically to the PIC, as expected, but CKM did not bind, indicating that CKM was excluded from the PIC–cMed complex. This was true irrespective of whether CKM(A) alone, CKM(A) in the presence of ATP, or CKM(KD) in the presence of ATP was used. This demonstrates that CKM exclusion from the PIC–cMed complex is predominantly a steric effect, rather than an effect mediated by Cdk8 kinase activity. To corroborate these findings, we performed an in vitro competition assay with purified CKM(KD), cMed, and pol II. In this assay, we took advantage of the maltose binding protein (MBP) tag on the CKM. After binding of the CKM to beads, overstoichiometric amounts of cMed were added to ensure saturated binding. The beads were then copiously washed to remove any unbound cMed. Finally, wash buffer with increasing concentrations of pol II was added, and the washes were analyzed by Western blot analysis using an antibody against Med17, one of the subunits of cMed. We observed the presence of increasing amounts of Med17 with increasingly added pol II (Fig. 2B). This experiment showed that pol II competes with the CKM for cMed binding and implicated pol II as the PIC component responsible for preventing the incorporation of the CKM into the cMed–PIC complex. Thus, we provide direct biochemical evidence that CKM–cMed and PIC–cMed are mutually exclusive complexes. To investigate where on the cMed surface CKM binds, we subjected the CKM–cMed complex to chemical crosslinking coupled to MS (XL–MS). CKM and cMed were mixed at a 1:1 ratio, dialyzed into lower salt, and then crosslinked with 1 mM bis(sulfo)succinimidyl suberate (BS3), after having titrated the crosslinker concentration to a concentration just below complete crosslinking (see Experimental procedures section). The crosslinked complex was then analyzed on a native gel to separate the CKM–cMed complex from the two-component subcomplexes. CKM–cMed was extracted from the gel, trypsin digested, enriched for crosslinked peptides, and subjected to mass spectrometric analysis. The analysis was carried out with a false discovery rate (FDR) cutoff of 1%. The obtained crosslinking network (Fig. 3A) showed not only extensive crosslinking within the CKM and cMed subcomplexes but also several high confidence crosslinks between CKM and cMed. To validate the interaction map, crosslinks between cMed subunits, for which the structure is already known, were mapped onto the structure (Fig. S2C). Crosslinks used for validation are shown on the interaction map in black, together with the measured atomic distances (Fig. S2C). The appearance of many crosslinks within the expected distance constraints confirmed the validity of the crosslinking network. The high confidence crosslinks between the CKM and cMed were mapped onto the cMed structure and are indicated by red spheres and corresponding red lines on the interaction network (Fig. 3B). Crosslinks that could not be mapped because of their presence in flexible regions were assigned to the closest structured residue and are indicated by pink spheres and corresponding pink lines on the interaction network. Thus, pink spheres indicate approximate crosslinking locations, and red spheres indicate exact locations. Dashed lines connected to the spheres indicate the complementary residues on the CKM. Crosslinks for which more than one unique match was found are emphasized in bold face. We found that the vast majority of crosslinks between the CKM and cMed fall on the pol II-binding face of cMed, with a larger number of crosslinks to the cMed middle module than to the head. In particular, a clustering of crosslinking sites to the CKM subunits Med12 and Med13 is seen on the knob and hook domains of cMed. On the other hand, Cdk8 and CycC crosslink to the head module spine and moveable jaw, respectively. These results show that CKM binds on one side of cMed, the side that faces pol II in the PIC–cMed complex, suggesting that CKM and pol II sterically exclude each other in cMed binding. In Figure 3C, the pol II–cMed interaction within the PIC is shown. Pol II is rendered with low opacity to allow seeing through pol II into the pol II–cMed interface. The presence of crosslinks directly on, and in close spatial proximity to, this interface, suggests that pol II and the CKM share an overlapping binding surface on cMed, indicating that CKM sterically excludes cMed from binding the PIC. We next investigated the role of the kinase activity of the CKM. We set up a series of exploratory studies to identify novel phosphorylation targets of the CKM within transcription initiation complexes. We used phosphopeptide enrichment and MS to map and compare phosphorylation sites on CKM(KD) and CKM(A). We found that the CKM undergoes extensive intra-CKM phosphorylation in vitro. We mapped these sites onto the CKM subunits (Fig. 4A). Phosphorylation sites found only in the CKM(A) sample, but not in the CKM(KD) sample, and therefore attributed to intra-CKM phosphorylation by Cdk8, are represented in orange. Phosphorylation sites found on the CKM(KD) sample are represented in teal and are likely deposited by the insect cell expression host as they appear on both CKM(A) and CKM(KD). A large number of intra-CKM phosphorylation sites occur on the ∼500-residue unstructured insertion in the Med13 subunit (Fig. 4A). This insertion is conserved from yeast to human and is located near the interface involved in binding cMed (41Tsai K.L. Sato S. Tomomori-Sato C. Conaway R.C. Conaway J.W. Asturias F.J. A conserved mediator-CDK8 kinase module association regulates mediator-RNA polymerase II interaction.Nat. Struct. Mol. Biol. 2013; 20: 611-619Crossr
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